Many bioactive metabolites possess unusual carbohydrates required for molecular recognition. (See for example, Liu, H.-w.; Thorson, J. S. Ann. Rev. Microbiol., 1994, 48, 223–256; Weymouth-Wilson, A. C. Nat. Prod. Rep. 1997, 14, 99–110; In Macrolide Antibiotics, Chemistry, Biology and Practice; Omura, S. Ed., Academic Press: New York; 1984; Johnson, D. A.; Liu, H.-w. Curr. Opin. Chem. Biol. 1998, 2, 642–649; and Trefzer, A.; Salas, J. A.; Bechthold, A. Nat. Prod. Rep. 1999, 16, 283–299.) In fact, roughly 70% of current lead compounds in modern drug discovery derive directly from natural products, many of which are glycosylated metabolites. (See Thorson, J. S. et al. Nature's Carbohydrate Chemists: The Enzymatic Glycosylation of Bioactive Bacterial Metabolites. Curr. Org. Chem. manuscript in press, (2000); and references therein and Weymouth-Wilson, A. C. The Role of Carbohydrates in Biologically Active Natural Products. Nat. Prod. Rep. 14, 99–110 (1997)). Examples of pharmaceutically important glycosylated metabolites include, for example, amphotericin, megalomicin/erythromycin, mithramycin, doxorubicin, vancomycin and calicheamicin, as shown in FIG. 5. While it is known that the sugar moieties of these pharmaceutically important metabolites often define their corresponding biological activity, (see Weymouth-Wilson, A. C., The Role of Carbohydrates in Biologically Active Natural Products, Nat. Prod. Rep. 14, 99–110 (1997)), efficient methods to systematically alter these essential carbohydrate ligands are still lacking.
In metabolite biosynthesis, glycosylation begins with the nucleotidylyltransferase-catalyzed activation of a sugar phosphate as a nucleotide diphosphosugar (NDP-sugar) donor. After activation, a number of enzymatic processing reactions often occur (e.g., deoxygenation, transamination, oxidation/reduction, epimerization, alkylation, and decarboxylation) prior to the culminating glycosyltransferase-catalyzed attachment to the aglycon. (Liu, H.-w. & Thorson, J. S. Pathways and Mechanisms in the Biogenesis of Novel Deoxysugars by Bacteria. Ann. Rev. Microbiol. 48, 223–256 (1994); Kirschning, A., Bechtold, A. F-W. & Rohr, J. Chemical and Biochemical Aspects of Deoxysugars and Deoxysugar Oligosaccharides. Top. Curr. Chem. 188, 1–84 (1997); Johnson, D. A. & Liu, H.-w. Mechanisms and Pathways from Recent Deoxysugar Biosynthesis Research. Curr. Opin. Chem. Biol. 2, 642–649 (1998); Hallis, T. M. & Liu, H.-w. Learning Nature's Strategies for Making Deoxy Sugars: Pathways, Mechanisms, and Combinatorial Applications. Acc. Chem. Res. 32, 579–588 (1999); Johnson, D. A. & Liu, H.-w. In Comprehensive Chemistry of Natural Product Chemistry (Barton, D.; Nakanishi; K.; Meth-Cohn, O. eds), Elsevier Science, Oxford, 311, (1999); Trefzer, A., Salas, J. & Bechthold, A. Genes and Enzymes Involved in Deoxysugar Biosynthesis in Bacteria. Nat. Prod. Rep. 16, 283–299 (1999); and Bechthold, A. & Rohr, J. In New Aspects of Bioorganic Chemistry (Diederichsen, U.; Lindhorst, T. K.; Wessjohann, L.; Westerman, B., eds.) Wiley-VCH, Weinheim, 313, (1999)).
The glycosyltransferases that incorporate these essential ligands are thought to rely almost exclusively upon UDP- and TDP-nucleotide sugars; however some have demonstrated promiscuity towards the sugar donor, (e.g., Gal, D-galactose; Glc, D-glucose; Man, D-mannose; NTP, nucleotide triphosphate; pFPTC, pentafluorophenoxythiocarbonyl; TDP, thymidine diphosphate; TMP, thymidine monophosphate; TTP, thymidine triphosphate; UDP, uridine diphosphate.) Genetic experiments suggest that downstream glycosyltransferases in secondary metabolism are promiscuous with respect to their NDP-sugar donor, setting the stage for the expansion of “combinatorial biosynthesis” approaches to change metabolite glycosylation. (See Madduri, K. et al., Production of the antitumor drug epirubicin (4′-epidoxorubicin) and its precursor by a genetically engineered strain of Streptomyces peucetius Nat. Biotech. 16, 69–74 (1998); and Hutchinson, C. R. Combinatorial Biosynthesis for New Drug Discovery. Curr. Opin. Microbiol. 1, 319–329 (1998).) This information has led to the exploitation of the carbohydrate biosynthetic machinery to manipulate metabolite glycosylation, (Madduri, K.; Kennedy, J.; Rivola, G.; Inventi-Solari, A.; Filppini, S.; Sanuso, G.; Colombo, A. L.; Gewain, K. M.; Occi, J. L.; MacNeil, D. J.; Hutchinson, C. R. Nature Biotech. 1998, 16, 69–74; and Zhao, L.; Ahlert, J.; Xue, Y.; Thorson, J. S.; Sherman, D. H.; Liu, H.-w. J. Am. Chem. Soc., 1999, 121, 9881–9882 and references therein), revitalizing interest in methods to expand the repertoire of available UDP- and TDP-sugar nucleotides. (See Zhao, Y.; Thorson, J. S. J. Org. Chem. 1998, 63, 7568–7572; and Elhalabi, J. M.; Rice, K. G. Cur. Med. Chem. 1999, 6, 93–116.)
These in vivo methods are limited by both a particular host's biosynthetic machinery and the specific host's tolerance to each newly constructed metabolite. Further, in vitro progress in this area is limited by the availability of the required NDP-sugar substrates. (Solenberg, P. J. et al., Production of Hybrid Glycopeptide Antibiotics in vitro and in Streptomyces toyocaensis. Chem. & Biol. 4, 195–202 (1997).)
Thus, there is a need for a greater variety of available NDP-sugar substrates.
Salmonella enterica LT2 α-D-glucopyranosyl phosphate thymidylyltransferase (Ep) is a member of the prevalent nucleotidylyltransferase family responsible for the reversible conversion of α-D-hexopyranosyl phosphate and NTP to the corresponding NDP-sugar nucleotide and pyrophosphate. Of the many nucleotidylyl-transferases studied, the NDP-sugar nucleotide-forming thymidylyltransferases have received the least attention in prior work. (See Lindquist, L.; Kaiser, R.; Reeves, P. R.; Lindberg, A. A. Eur. J. Biochem. 1993, 211, 763–770, and Gallo, M. A.; Ward J.; Hutchinson, C. R. Microbiol. 1996, 142, 269–275.) Even in Ep, substrate specificity studies prior to the work of the present inventors were limited to only a few available hexopyranosyl phosphates. (See Lindquist, L.; Kaiser, R.; Reeves, P. R.; Lindberg, A. A. Eur. J. Biochem. 1993, 211, 763–770.)